Alternative cryoprotective agent for corneal stroma-derived mesenchymal stromal cells for clinical applications

Cryopreservation of human corneal stroma-derived mesenchymal stromal cells (hCS-MSCs) with dimethylsulfoxide (DMSO) as a cryoprotective agent (CPA) has not been previously compared to that with glycerol under standard conditions. The hCS-MSCs were hereby cryopreserved with both compounds using a freezing rate of 1 °C/minute. The CPAs were tested by different concentrations in complete Minimum Essential Medium (MEM) approved for good manufacturing practice, and a medium frequently used in cell laboratory culturing—Dulbecco’s modified eagle serum. The hCS-MSCs were isolated from cadaveric human corneas obtained from the Norwegian Eye Bank, and immunophenotypically characterized by flow cytometry before and after cryopreservation. The survival rate, the cellular adhesion, proliferation and cell surface coverage after cryopreservation of hCS-MSCs has been studied. The hCS-MSCs were immunofluorescent stained and examined for their morphology microscopically. The results showed that cryopreservation of hCS-MSCs in MEM with 10% glycerol gives a higher proliferation rate compared to other cryopreserving media tested. Based on the results, hCS-MSCs can safely be cryopreserved using glycerol instead of the traditional use of DMSO.


Cell adhesion
The adhesion of hCS-MSCs after thawing them showed certain variability between the different CPAs.The hCS-MSCs cryopreserved with 10% glycerol in complete DMEM adhered to the plastic culture plate with a significantly higher number of cells in comparison to the cells cryopreserved with DMSO (Fig. 2C).Additionally, the cells cryopreserved with 15% glycerol adhered with a significantly higher number than the cells cryopreserved with 20% glycerol (Fig. 2C).

Cell surface covering, cell spreading and image analysis
The hCS-MSCs plastic culture surface coverage in the T25 flask during the 4 days after thawing the cells cryopreserved in different CPAs and media (complete MEM or DMEM) showed that the cells were able to grow and cover the surface in the flask in an increasing manner over the observation period (Figs. 4, 5, 6).
The hCS-MSCs cryopreserved with any CPA in complete DMEM showed 20-60% of surface coverage at Day 3 and 4 (Fig. 4B), while the cells cryopreserved with any CPA in complete MEM showed values around 20% on Day 4 (Fig. 4C).The hCS-MSCs in complete DMEM were able to cover a larger surface area than those in complete MEM within the first 4 days after thawing (Fig. 4).The hCS-MSCs cryopreserved with DMSO and 10% glycerol in DMEM had a significant increase in surface coverage from Day 1 to Day 4 (Fig. 4B).
The immunofluorescent stainings and the brightfield images presented here on different days of growth after thawing the hCS-MSCs, showed that the cells have a fibroblastoid morphology (Figs. 4 and 5).Analysis of the immunofluorescent stainings of the hCS-MSCs at the day 0 after seeding them cryopreserved with any CPA in complete DMEM or MEM showed cells with a cellular area of approximately 1500 µm 2 (Fig. 5).In addition, the cells cryopreserved with 10% DMSO in complete DMEM proved to be significantly bigger than those cryopreserved with 20% glycerol, while the hCS-MSCs cryopreserved with 10% glycerol were significantly more spread than the cells cryopreserved in 15% and 20% glycerol (Fig. 5A).Furthermore, the hCS-MSCs cryopreserved in complete DMEM or MEM supplemented with 10% glycerol were able to differentiate towards chondrogenic, adipogenic and osteogenic lineages (See Supplementary Fig. S2).
The data obtained with the brightfield images during the 4 days of experiments showed similar results (Fig. 6).The hCS-MSCs preserved with any CPA in complete DMEM demonstrated to have cell surface area values of approx.2000 µm 2 from Day 1 to 4 (Fig. 6A), and the hCS-MSCs cryopreserved with 10% DMSO or 10% glycerol www.nature.com/scientificreports/ in complete DMEM showed a significantly higher cell surface area coverage than hCS-MSCs cryopreserved with 15% glycerol in complete DMEM at Day 3 (Fig. 6A).Remarkably, the hCS-MSCs cell surface area with any of the CPAs in complete DMEM at any time-point showed to be approximately 500 µm 2 higher than hCS-MSCs with any CPA in complete MEM at any time point (Fig. 6).In complete MEM, the hCS-MSCs preserved with any CPA appeared to have a cell surface area coverage of approximately 1500 µm 2 at any time-point (Fig. 6B).

Discussion
It is known that the use of CPAs for long-term storage of cells is important to preserve the immunophenotypical characteristics of the cells, their viability and ability to proliferate 26 .Cryopreservation of cells is an important stress factor that can affect the permeabilization of their cell membrane 26,27 .Nowadays, there are many alternative CPAs and methodologies to preserve cells, and it is crucial to adjust the CPA methodologies according to the cell type that is being used 20 .In the present study, glycerol was used as an alternative CPA to preserve hCS-MSCs, and it was compared to the gold standard (DMSO).The International Society for Cellular Therapy has proposed minimal criteria for defining MSCs 28,29 .These parameters are based on plastic culture plate adherence and ≥ 95% of the MSC population expressing CD73, CD90 and CD105, while ≤ 2% should express CD34, CD45, CD11b or CD14, CD79 or CD19 and HLA class II 28,29 .Likewise, the multipotent differentiation of the cells needs to be demonstrated by immunostaining in vitro 28,29 .The immunophenotype characterization of hCS-MSCs cryopreserved with 10% DMSO or 10% glycerol in complete DMEM and complete MEM showed that hCS-MSCs possess the main characteristics of MSCs, which have been described previously by us and others 7,30 .However, it was possible to observe certain differences between the cells preserved with 10% DMSO and 10% glycerol in complete MEM media, and 10% glycerol in complete DMEM media when compared to the values obtained by Vereb et al. 7 .The hCS-MSCs cryopreserved with both 10% DMSO and 10% glycerol in complete MEM media expressed CD146 values closer to the ones found in the literature 7 .Additionally, the presence of CD117 (an early progenitor marker) increased on hCS-MSCs cryopreserved with 10% glycerol in complete MEM, reaching similar values to those found in the literature 7 .The hCS-MSCs cryopreserved with 10% DMSO and 10% glycerol in complete MEM media showed a significant increase in CD184/CXCR4 reaching values of 16% and 97%, respectively.This finding www.nature.com/scientificreports/somewhat differs from the data presented by the researchers who described these cells for the first time 7 .Nevertheless, CD184/CXCR4 is known to disappear during cell isolation and subculturing, and some authors have demonstrated a later recovery of this chemokine receptor 31 .CD184/CXCR4 is an important marker involved in many cellular conditions and types of cells, and it is expressed in stem cells and cancer cells 32,33 .This marker has also been shown to have an important role in tissue regeneration (e.g.nervous tissue, heart, lung and liver) 34 , and it mediates progenitor cell homing and recruitment to injury sites, being involved in cell arrest, survival and angiogenesis 35 .Therefore, the indicated effects of CD184/CXCR4 and the ligand CXCL12 can be considered valuable when discovering the characteristics of hCS-MSCs cryopreserved in complete MEM media and more significantly, glycerol.The presented results indicate that glycerol as a CPA could be a better cryoprotectant than the gold standard (DMSO) for hCS-MSCs, the latter previously being described as the best cryoprotectant by many authors 26,[36][37][38][39][40][41][42] .
The survival rate of the hCS-MSCs cryopreserved with the different CPAs in complete DMEM or complete MEM showed no statistical differences with an approximate loss of 20% of the viable cells, in line with the described loss of viable cells after cryopreservation in the literature, depending on the cell type and the CPA used 43 .Several authors have found a higher loss of cells (30% viable cells) 44 , while others have shown a survival rate of approximately 80-90% 45,46 .It is known that the survival rate may vary depending on the cryopreservation method being used 47 .
The cell adhesion ability of the cryopreserved hCS-MSCs in the different CPAs in complete DMEM or MEM showed that the cells preserved with 10% glycerol have better adhesion qualities than those frozen with 10% DMSO.Other have shown similar findings, observing that MSCs have better attachment qualities after preserving them in alternative CPAs other than DMSO 48 .This supports the hypothesis that 10% glycerol could be a better CPA than DMSO for hCS-MSCs.
There were no differences in the proliferation of the hCS-MSCs with any of the CPAs in complete DMEM, but in line with the previous findings, hCS-MSCs preserved with 10% glycerol in complete MEM demonstrated a significantly better proliferation compared to those cryopreserved with DMSO.Human platelet lysate (HPL) can replace FBS for culturing human MSCs showing better capabilities such as increased cell proliferation, differentiation potential, without affecting the MSC immunophenotype, and potential of immunomodulation among other important factors 12,49,50 .The use of HPL, in addition, helps to reach GMP grade in cryopreservation 12,51 .
The fibroblastoid characteristics of the hCS-MSCs are in line with the fact that these cells can migrate to a damaged area 52,53 .When hCS-MSCs are intact in the cornea, the cells present a dendritic morphology and www.nature.com/scientificreports/low proliferation rate 54,55 .When the cornea suffers an injury, the hCS-MSCs attain a fibroblastic morphology, analogous to the hCS-MSCs being cultured in vitro 52,53 .
The hCS-MSCs stored with 20% glycerol presented a spheroid morphology more often than the other glycerol concentrations and DMSO.Although unclear why, it is likely the higher concentration of glycerol could be a key factor contributing to such a morphology 25 .The high viscosity of the glycerol could promote such differences in morphology as seen with other viscous solutions 56 among other effects 25 .As hCS-MSCs are stromal cells, one should consider higher cell spreading as a beneficial quality, since stromal cells are involved in maintaining the structural integrity of the cornea and the healing of damaged tissue 57 .
The hCS-MSCs appeared more spread in complete DMEM compared to in complete MEM, and the cells preserved with 10% DMSO and 10% glycerol most extensive spreading among all tested conditions.This fact, in addition to the higher proliferation of hCS-MSCs in presence of HPL in complete MEM, could also suggest that hCS-MSCs have a spheroid morphology due to the rapid growth of the cells.Moreover, a higher proliferation rate, could mean hCS-MSCs are dedicating more energy to their reproduction, copying the DNA and other cellular processes related to cells' division, rather than employing energy on spreading and attaching to a larger surface 58 .
Altogether, the present study shows that we could successfully seek and test an alternative CPA for hCS-MSCs in complete DMEM or MEM.The preservation of hCS-MSCs with 10% glycerol in complete DMEM or MEM demonstrated a very similar survival rate, surface coverage and cell area compared to the hCS-MSCs cryopreserved by 10% DMSO in complete MEM or DMEM.The hCS-MSCs cryopreserved in complete DMEM could cover a larger cell surface area than cells in complete MEMThis can be beneficial in terms of the function of any stromal cells, as well as their spreading and generation of extracellular matrix.The larger size of the cells could be a consequence of their slower reproduction, and further research is required to better understand why the cells present such a morphology in complete DMEM instead of complete MEM.MSCs have a variable growth depending on the composition of the media and the presence of human-derived cell media components instead of components derived from animals 12,51 .Despite these facts, hCS-MSCs cryopreserved with 10% glycerol in complete DMEM or MEM presented similar capabilities compared to those stored with the gold standard DMSO.Additionally, hCS-MSCs cryopreserved by 10% glycerol in complete MEM demonstrated a better cellular adhesion, and in some cases better cell proliferation and recovery of the migratory marker (CD184/CXCR4) compared to cells cryopreserved with 10% DMSO.The cryopreservation with 10% glycerol, independent of the media chosen, demonstrated to be a non-toxic CPA alternative that could be utilized to preserve hCS-MSCs, and eventually be used to store these cells for future cell-therapy following proper GMP guidelines.
Other cells types need to be tested and validated under the same cryopreserving conditions in separate experiments to show general applicability of our findings.

Isolation and cultivation of cells
The hCS-MSCs were isolated from cadaver eyes obtained as leftover material from Norwegian Eye Bank (following DSAEK operation).The processing and use of the human tissue are in accordance with the directives of the Helsinki Declaration and all tissue harvesting was approved by the Regional Committee for Medical and Health Research Ethics (REK 2017/418) in Norway.The hCS-MSCs isolation was performed following our own methodology described by Nagymihaly et al. 59 .Briefly, the corneal disc was separated from the corneal scleral ring, placed in a Petri dish with Dulbecco's Phosphate Buffered Saline (DPBS) 1X, and the epithelial and Descemet's membranes were removed by scratching under a stereomicroscope, rinsed and cut in approximately 10-12 pieces.The tissue pieces were then transferred to a microplate of 6 wells (Corning ® , Axygen ® , Merck/ Sigma-Aldrich, MO, USA) placing 1 or 2 pieces in each well with 800 µL of Dulbecco's modified eagle medium (DMEM) low glucose with Glutamax™ supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco ® , Thermo Fisher Scientific, MA, USA) and 1% antibiotic/antimycotic solution (Merck/Sigma-Aldrich, MO, USA) (complete DMEM) and incubated at 37 °C 5% CO 2 for 24 h.After the first day in culture, the wells were carefully filled up with 2-3 mL of pre-warmed complete DMEM, and the culture media was changed every 3-4 days.The adherent hCS-MSCs from the attached tissue pieces were trypsinized, filtered with a strainer with at least 70 µm pore size, and cryopreserved in liquid nitrogen at -196 °C until further use, approximately 25-30 days after the isolation day.The isolated hCS-MSCs were phenotypically characterized by flow cytometry using a Becton Dickinson (BD) FACS Canto II (BD biosciences, USA) and BD Stemflow Human MSC Analysis Kit (BD biosciences, USA) prior to the experiments.

Storage of hCS-MSCs by applying different cryoprotective agents
The hCS-MSCs were frozen using two different CPAs: standard 10% DMSO (Merck/Sigma-Aldrich, MO, USA) in complete DMEM or MEM, and glycerol (Merck/Sigma-Aldrich, MO, USA) at three different concentrations (10, 15, and 20%) in complete DMEM or MEM (see Table 1 for the experimental setup of the media used).
Three primary hCS-MSCs donors were counted with a Bürker chamber using Tryphan Blue to visualize the live cells and, subsequently, approximately 7.5•10

Quantification of hCS-MSCs
The hCS-MSCs cryopreserved with the different CPAs were rapidly thawed in a water bath after 2-4 weeks in liquid nitrogen and counted using a Bürker chamber by Trypan blue to calculate the survival rate ((cells alive after thawing/cells alive before freezing) × 100).Subsequently, the live cells were transferred into 48 well plates to study the cell adhesion after 6 h incubation, and the proliferation assay from day 1 to day 4.At the end of each timepoint, the hCS-MSCs were rinsed twice with DPBS (1X) and lysed by 300 μL of mammalian-protein extraction reagent (Thermo Fisher Scientific, MA, USA).Once all samples were collected, the relative number of attached cells was quantified by measuring the lactate dehydrogenase (LDH) activity using the Cytotoxicity Detection KitPLUS LDH (Roche Applied Science, Penzberg, Germany).The samples were measured spectrophotometrically at 492 nm using a Victor 3 microplate reader (PerkinElmer, Waltham, MA, USA), and the number of cells were calculated using the calibration curve prepared with known cell numbers.All the experimental conditions were performed in triplicate.

Human CS-MSCs trilineage differentiation
Human CS-MSCs cryopreserved with 10% glycerol were differentiated to three lineages using adipogenesis, chondrogenesis and osteogenesis kits (Gibco, Stem-Pro, Thermo Fisher Scientific, MA, USA).The kit media were replaced every 3-4 days for a period of 2-3 weeks in a 48-well plate.Once the differentiation period ended, the media were removed and the cells were fixated in 4% formalin solution.Alizarin Red S (Merck/Sigma-Aldrich), Alcian Blue solution (Merck/Sigma-Aldrich), and HCS LipidTOX (Invitrogen, Thermo Fisher Scientific, MA, USA) were used to stain fixated hCS-MSCs for osteogenesis, chondrogenesis, and adipogenesis, respectively.Images were taken using an EVOS FL fluorescent microscope or a Zeiss upright light microscope.

Cell surface covering, cell spreading and image analysis
In parallel to the cell adhesion and proliferation assays, 50 000 cells were placed in culture in T-25 flasks to quantify the surface coverage at days 1, 2, 3 and 4 through brightfield microscopy images.Moreover, 20 000 cells were seeded in duplicates in a 48 well plate, and after 6 h incubation, the attached hCS-MSCs were rinsed twice with DPBS (1X), fixed with 10% formalin for 10 min, washed 3 times, and the cells were stained with Vimentin (MA5-16,409, Rabbit Monoclonal, 1:200, Thermo Fisher Scientific, MA, USA) and DAPI (D1306, dilution 1:1000, Thermo Fisher Scientific, MA, USA).Images were taken by an EVOS FL fluorescent microscope (Thermo Fisher Scientific, MA, USA), and further analyzed using ImageJ 1.51w software (NIH, Bethesda, MD, USA) to determine cell area and surface coverage.

Figure 2 .
Figure 2. Survival studies and cell adhesion of hCS-MSCs under different conditions.(A,B) hCS-MSCs survival percentage in (A) complete DMEM and (B) complete MEM media after cryopreservation with different cryoprotective agents (10% DMSO, 10, 15 and 20% glycerol).(C,D) Cell adhesion to plastic surface after 6 h of incubation post-thawing (C) in complete DMEM and (D) complete MEM media (GMP media).All data expressed as average ± SD of 3 primary hCS-MSCs lines, (*p < 0.05).
5-1•10 6 cells/mL were cryopreserved in media containing DMSO or glycerol.The hCS-MSCs cryopreserved in DMSO were gently mixed and immediately transferred into − 80 °C inside a freezing container (Mr. Frosty; 1 °C/min cooling rate), while the hCS-MSCs cryopreserved with glycerol were gently mixed in the cryovial and left on the bench for 20 min prior to transferring into − 80 °C inside the Mr. Frosty.All the cryovials were transferred after a few days into liquid nitrogen for at least two weeks storage.

Table 1 .
Different cell culturing media and cryoprotective agents (CPAs) used in the experiments.